The ERV-9 LTRs of human endogenous retroviruses are middle repetitive DNAs that contain unusual sequence features in the U3 enhancer region. The broad long-term objective is to ascertain the functional significance of the ERV-9 LTRs dispersed in the human genome in transcriptionally activating and thus marking the cis-linked loci of hematopoietic genes and gene families in early progenitor cells during ontogeny and hematopoietic lineage differentiation. The specific aim is to test the hypothesis that the solo ERV-9 LTR located near the HS5 site in the 5' border of the human beta-globin locus control region (LCR) initiates transcription of the LCR during early stages of ontogeny and that this transcription process of the LCR regulates the transcriptional activation of the further downstream beta-like globin genes during erythropoiesis. Specific aspects of this hypothesis will be tested. 1). The U3 enhancer repeats of the 5'HS5 LTR may bind to a limited set of cognate transcription factors abundantly expressed in erythroid and placental trophoblast cells. These transcription factors will be identified by electrophoretic mobility shift assays. Genes encoding new transcription factors will be cloned. The molecular architecture and function of the U3 enhancer complex will be examined by site-directed mutagenesis of the U3 repeats in test plasmids containing the Green Fluorescent Protein reporter gene and by transfection assays. 2). Replacement of the 5'HS5 LTR with an LTR from a different family of human endogenous retroviruses disrupts the transcription of the beta-globin LCR and the transcriptional activation of the further downstream human beta-like globin genes in a YAC clone spanning the entire human beta-globin gene locus. Transgenic mice harboring the mutant YAC clones will be created for this study. 3). A functionally equivalent solo LTR of a murine endogenous retrovirus may exist in the 5' boundary area of the mouse beta- globin LCR. The murine 5' boundary area of the beta-globin LCR cloned in a BAC vector will be sequenced in an attempt to identify a murine LTR with similar sequence features and in vitro functional characteristics as the human 5'HS5 LTR. The proposed work may provide information on the functional organization of the human genome and aid in the effort on gene therapy of hereditary human diseases.